The Arabidopsis DNA glycosylase MBD4L repairs the nuclear genome in vivo.

SUMMARY: DNA glycosylases remove mispaired or modified bases from DNA initiating the base excision repair (BER) pathway. The DNA glycosylase MBD4 (methyl‐CpG‐binding domain protein 4) has been functionally characterized in mammals, but not yet in plants, where it is called MBD4‐like (MBD4L). Mammalian MBD4 and Arabidopsis recombinant MBD4L excise U and T mispaired with G, as well as 5‐fluorouracil (5‐FU) and 5‐bromouracil (5‐BrU) in vitro. Here, we investigate the ability of Arabidopsis MBD4L to remove some of these substrates from the nuclear genome in vivo in coordination with uracil DNA glycosylase (AtUNG). We found that mbd4l mutants are hypersensitive to 5‐FU and 5‐BrU, as they displayed smaller size, less root growth, and higher cell death than control plants in both media. Using comet assays, we determined BER‐associated DNA fragmentation in isolated nuclei and observed reduced DNA breaks in mbd4l plants under both conditions, but particularly with 5‐BrU. The use of ung and ung x mbd4l mutants in these assays indicated that both MBD4L and AtUNG trigger nuclear DNA fragmentation in response to 5‐FU. Consistently, we here report the nuclear localization of AtUNG based on the expression of AtUNG‐GFP/RFP constructs in transgenic plants. Interestingly, MBD4L and AtUNG are transcriptionally coordinated but display not completely overlapping functions. MBD4L‐deficient plants showed reduced expression of BER genes and enhanced expression of DNA damage response (DDR) gene markers. Overall, our findings indicate that Arabidopsis MBD4L is critical for maintaining nuclear genome integrity and preventing cell death under genotoxic stress conditions. Significance Statement: MBD4 is a DNA glycosylase from the base excision repair (BER) system that is known to affect cell death, tumor progression, and gene expression in mammals, but has been poorly studied in plants. Our work provides solid evidence on the role of the Arabidopsis MBD4 enzyme (MBD4L) on nuclear genome repair in vivo; its activity on particular substrates; its differences with AtUNG; its coordination with other BER enzymes; and its effects on plant growth, cell viability, and responses to DNA damage. [ABSTRACT FROM AUTHOR]